The goals of this research application are a) to test the hypothesis that the variable human gamma-globin gene silencing in the adult is the consequence of a dynamic equilibrium between euchromatin originating in the LCR and heterochromatin originating in the gamma gene promoter, b) to identify gamma-globin gene specific repressors and corepressors. Our specific aims are i) To test the hypothesis that the gamma gene silencing in the adult is the consequence of a dynamic balance between euchromatin originating in the LCR and heterochromatin originating in the gamma gene promoter. This will be tested in transgenic mice carrying the human beta-globin locus yeast artificial chromosome (betaYAC) and various mutated betaYACs by examining changes of the histone code specific for heterochromatin and euchromatin. This hypothesis can be validated if changes of the histone code are correlated with the phenotypes induced by the various mutations, ii) To test whether the gammaCACCC box causes heterochromatinization in the gamma gene promoter in the adult. This will be done by relocating the gammaCACCC box in the different locations in the beta-globin locus and examining formation of heterochromatin induced by the gammaCACCC box. iii) To develop an oligonucleotide-mediated chromatin immunoprecipitation approach and using this approach to search for gamma gene specific repressors/corepressors. It is expected that these studies will lead to a unifying model explaining variable silencing of human gamma-globin gene in the adult, and will identify gamma gene specific repressors/corepressors. These studies will facilitate designing of a feasible strategy for human gamma-globin gene reactivation. Such a development will have important consequences for the treatment of patients with sickle cell disease or beta thalassemia syndromes.